In the present study, RA SF contained greater amounts of IP-10 as compared with OA SF. Immunolocalization analysis indicated that IP-10 was associated mainly with infiltrating macrophage-like cells, and fibroblast-like cells in the RA synovium, as described previously [25
]. In addition, substantial amounts of IP-10 were also secreted from RA SF monocytes in vitro
and, to a lesser extent, from RA SF PMNs cocultured with FLSs. The present study clearly demonstrates that cell–cell interactions that occur in the RA joint tissues are important for induction of IP-10 expression. The augmentation of IP-10 production was dependent on an interaction between synovial FLSs and leukocytes; individually, none of the cell populations tested produced substantial amounts of IP-10. Indeed, the necessity for physical contact between the cells was apparent from the finding that IP-10 production was completely blocked by a transwell membrane that separated FLSs from the leukocytes, but was permeable to soluble factors.
The pathway governing IP-10 expression was further examined by determining the role of adhesion molecules in the regulation of IP-10 production mediated by FLS–leukocyte interactions. Application of neutralizing anti-CD11b, CD18, or anti-ICAM-1 monoclonal antibodies to FLS–leukocyte cocultures significantly inhibited IP-10 production (Fig. ). This implies that upregulation of IP-10 production by cell–cell contact was, in large part, promoted through a β2-integrin/ICAM-1-mediated mechanism, although it remains to be tested whether other adhesion molecules are involved in the induction of IP-10 mediated by the interaction of RA FLSs and leukocytes. This pathway cannot solely account for the response, however, because monoclonal antibodies against either β2-integrin or ICAM-1 inhibited IP-10 secretion by, at most, 53–59% in FLS–monocyte coculture and by 54–87% in FLS–PMN coculture.
In addition, the findings presented here reveal that IP-10-inducible soluble factors, such as IFN-γ and TNF-α, which may be induced by cell–cell interactions, were not involved in IP-10 induction in this system, because we failed to detect significant inhibitory effects of anti-IFN-γ or anti-TNF-α antibodies on IP-10 secretion. Furthermore, we recently demonstrated that the secretion of a potent angiogenic factor, namely vascular endothelial growth factor, was markedly induced by the interaction of FLS with synovial leukocytes via the integrin/ICAM-1 pathway [19
]. Taken together, these data support the notion that the physical contact between either SF monocytes or neutrophils and FLSs might be important for producing inflammatory mediators, such as IP-10 or vascular endothelial growth factor, as is observed in the synovium of RA, and is further implicated in the progression of RA.
Additionally, IP-10 was originally found to be expressed and secreted by monocytes, fibroblasts, and endothelial cells after stimulation with IFN-γ [5
]. The present data clearly demonstrate that activated PMNs interacting with fibroblasts are an important cellular source of IP-10 in RA synovitis, because most of the leukocytes infiltrating the SF of rheumatoid joints are PMNs. PMNs in the RA SF are in an activated state, and produce a variety of other inflammatory mediators [22
]. Furthermore, neutrophils are recognized as an important cellular source of IP-10 [34
]. This biosynthetically active leukocyte population almost certainly contributes significantly to the disease process during active RA.
Th1 cells and Th1-type cytokines play an important role in the development of progressive synovitis in RA [13
]. CXCR3, a specific IP-10 receptor, is expressed preferentially in Th1 as compared with Th2 cells, and Th1 but not Th2 cells respond to IP-10 [36
]. Indeed, there are CXCR3-positive cells in RA synovium [25
]. Findings from those studies, together with the present data, support the hypothesis that IP-10 secreted by activated SF leukocytes interacting with fibroblasts might contribute to migration of Th1 cells through CXCR3 in the development of RA.